WO2013029025A1 - Compositions et méthodes permettant de moduler l'activité cellulaire - Google Patents

Compositions et méthodes permettant de moduler l'activité cellulaire Download PDF

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WO2013029025A1
WO2013029025A1 PCT/US2012/052391 US2012052391W WO2013029025A1 WO 2013029025 A1 WO2013029025 A1 WO 2013029025A1 US 2012052391 W US2012052391 W US 2012052391W WO 2013029025 A1 WO2013029025 A1 WO 2013029025A1
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nanoparticles
cells
cell
trpvl
nanoparticle
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Jeffrey Friedman
Sarah Stanley
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The Rockefeller University
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Priority to US14/239,427 priority Critical patent/US9399063B2/en
Publication of WO2013029025A1 publication Critical patent/WO2013029025A1/fr
Priority to US15/168,950 priority patent/US10064941B2/en
Priority to US16/049,102 priority patent/US10786570B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1611—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/40—Applying electric fields by inductive or capacitive coupling ; Applying radio-frequency signals
    • A61N1/403—Applying electric fields by inductive or capacitive coupling ; Applying radio-frequency signals for thermotherapy, e.g. hyperthermia
    • A61N1/406—Applying electric fields by inductive or capacitive coupling ; Applying radio-frequency signals for thermotherapy, e.g. hyperthermia using implantable thermoseeds or injected particles for localized hyperthermia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention provides methods and compositions for the remote control of cell function based on the use of radiofrequency waves to excite nanoparticles targeted to specific cell types.
  • the cell type of interest expresses a temperature sensitive channel wherein excitation of the nanoparticles results in a localized temperature increase that is transduced into a cellular response.
  • Such cellular responses may include, for example, increases in gene expression resulting in production of one or more physiologically active proteins.
  • the expression of such proteins can be used to treat a variety of different inherited or acquired diseases or disorders in a subject. Accordingly, the invention provides a generic approach for treatment of any disease associated with a protein deficiency.
  • ion channels has many advantages; their structure and function are relatively well described; they have a rapid time course of activation, and a broad range of channels exist in mammalian and non-mammalian cells, which may be exploited in the search for the optimum means of modifying cellular activity.
  • This approach was first validated by transgenic expression of a drug-gated channel to modify behavior, however, the time course of effects was relatively slow (hours to days) due to irreversible effects of the ligand.
  • the non-mammalian channelrhodopsin (ChR2) gene a light activated cation, has been employed to rapidly activate molecularly defined neurons when exposed to blue light (Boyden ES et al.
  • the present invention provides methods and compositions for the remote control of cell function based on the use of radiofrequency waves to excite nanoparticles targeted to specific cell types.
  • the invention uses Nanoparticle Induced Circuit Excitation (NICE) to, for example, regulate ion channels as a means for stimulating the activity of specific cells remotely and non-invasively.
  • NICE Nanoparticle Induced Circuit Excitation
  • the invention described herein utilizes Nanoparticle Induced Circuit Excitation (NICE), which encompasses compositions and methods that have been developed for stimulating the activity of specific cells remotely and non-invasively.
  • NICE Nanoparticle Induced Circuit Excitation
  • the present invention provides methods and compositions based on the use of radiofrequency waves to excite nanoparticles targeted to specific cell types.
  • the cell type of interest expresses a temperature sensitive channel wherein excitation of the nanoparticles results in a localized temperature increase that is transduced into a cellular response.
  • the excitation of the nanoparticles results in a localized temperature increase that is transduced into a cellular response such as, for example, an increase in gene expression.
  • Such increases in gene expression may result in production of one or more physiologically active proteins.
  • the described invention provides a method to remotely stimulate the activity of a cell type of interest wherein the nanoparticles are externally applied.
  • a method comprises: (i) administering to a cell population nanoparticles selective for the cell type of interest; and (ii) applying a radiofrequency field to remotely activate the nanoparticles. Said activation of the nanoparticles results in stimulation of the activity of the cell type of interest.
  • cells may be engineered to synthesize nanoparticles intracellularly.
  • the iron storage protein ferritin which forms a naturally occuring iron nanoparticle, was modified to form a ferritin fusion protein composed of a ferritin light chain fused to ferritin heavy chain with a flexible linker region. Heating of the iron core by a RF magnetic field opens the TRPVl channel to trigger calcium entry, increasing proinsulin gene expression and triggering insulin release in vitro. This results in decrease blood glucose in vivo.
  • modification using intracellular nanoparticles uses a modified TRPVl with a camelid antibody to GFP fused to the N-terminal of TRPVl and a modified ferritin fusion protein with EGFP fused to the N- terminal of ferritin light chain-linker-ferritin heavy chain. Heating of the iron core of the ferritin attached to the TRPVl triggers calcium entry and increases proinsulin gene expression and proinsulin release in vitro.
  • said nanoparticles may be paramagnetic nanoparticles.
  • a method to remotely stimulate the activity of a cell type of interest in a subject, the method comprising: (i) administering to the subject nanoparticles selective for the cell type of interest; and (ii) applying a radiofrequency field to remotely activate the nanoparticles. Said activation of the nanoparticles results in stimulation of the activity of the cell type of interest in a subject.
  • Activities of the cell that may be stimulated include, for example, cellular responses such as cell proliferation and/or differentiation, apoptosis, activation of signal transduction pathways, neuronal activation, development of long term potentiation and/or regulation of gene expression.
  • the invention provides a method to stimulate the activity of a cell type of interest in a subject, the method comprising steps: (a) administering to the subject modified cells of interest that comprise nanoparticles that are selective for the cell type of interest; and (b) applying a radiofrequency field to remotely activate the nanoparticles. Said activation of the nanoparticles results in stimulation of the activity of the cell type of interest in a subject.
  • the present invention can be used in a variety of different clinical settings.
  • the technology can be used to control the expression of physiologically active proteins for used in treatment of various inherited or acquired disorders or diseases.
  • stem cells such as induced pluripotent stem cells (iPSC) or autologous iPSC.
  • mesenchymal stem cells engineered to express NICE constructs could act as autografts enabling external control of cell function.
  • NICE dependent calcium entry can then be used to regulate functions including hormone release, muscle contraction, or neural activity.
  • Regulated hormone expression and release can facilitate the treatment of several endocrine conditions such as diabetes.
  • Neuronal stimulation can be used therapeutically in several debilitating conditions such as Parkinson's disease (subthalamic stimulation) and stroke (transcranial direct current stimulation), as well as for pain relief and gastroparesis (Benabid AL. et al, 2009 Lancet Neurol 8:67-81; Schlaug G. et al. 2008 Arch Neurol 65: 1571-1576; Nnoaham KE, Kumbang J 2008 Cochrane Database Syst RevCD003222; Maranki J, Parkman HP 2007 Curr Gastroenterol Rep 9:286-294).
  • Functional nanoparticles prepared using methods known to those skilled in the art, can be targeted by coating with recombinant antibodies directed to endogenous cell specific surface proteins. These applications and the approaches can be applied in animals using the NICE techniques.
  • the methods and compositions of the invention provide a means for dissecting the contributions of defined cell populations to physiology.
  • the present invention makes it possible to decorate different cell types with nanoparticles tuned to different frequencies, thus allowing one to simultaneously activate ensembles of defined cells even if they are in proximity.
  • the described invention provides for selective modification of cellular function non-invasively both in vitro and in vivo. At present, there are no methods for anatomically discrete, temporally controlled, non-invasive cell activation. Such a technique allows one to study the roles of cell populations in physiological processes, in particular those functions that are, or would be, perturbed by invasive methods.
  • the invention proves non-human transgenic animals containing different cell types that can be activated remotely through the targeting of nanoparticles to the surface of said cells.
  • the transgenic animals provide an in vivo means for studying the contributions of defined populations of cells to physiology.
  • the transgenic animals of the invention may be used as animal model systems for the screening, identification and testing of useful therapeutic compounds.
  • the described invention provides, for example, methods to remotely modulate cell function in vertebrates and apply NICE to (i) modify glucose metabolism (ii) activate dopaminergic neurons in the midbrain that control reward and (iii) use a combinatorial activation scheme to regulate feeding behavior.
  • a specific embodiment of the invention makes use of a unique combination of four components: (i) a radio frequency electromagnetic field; (ii) cell-specific expression of a nanoparticle tether; (iii) metallic/metal oxide nanoparticles; and (iv) a temperature sensitive TRPV cation channel to induce a tunable increase in intracellular calcium.
  • compositions comprising nanoparticles that are selective for a cell type expressing a temperature sensitive channel.
  • pharmaceutical compositions of the invention may comprise modified cells expressing a temperature sensitive channel of interest and decorated with nanoparticles selective for said cells.
  • FIGURE 1 Nanoparticles induced cell excitation to increase insulin expression and release in vitro. Schema of nanoparticle-induced cell activation and gene expression. Antibody-coated ferrous oxide nanoparticles bind to a unique epitope, His x 6, in the first extracellular loop of the temperature-sensitive TRPV1 channel. Exposure to a RF field induces local nanoparticle heating, which opens temperature-sensitive TRPV1 channels. Calcium entry triggers downstream pathways, such as activation of calcineurin, leading to dephosphorylation of NFAT and translocation to the nucleus.
  • NFAT binds to upstream response elements to initiate gene expression of a bioengineered human insulin gene.
  • Additional calcium-dependent signal transduction pathways also stimulate gene expression via binding to SRE and CRE.
  • P indicates a phosphate group.
  • FIGURE 2 Heating of iron oxide nanoparticles in RF magnetic field.
  • A Bulk heating effects of treating iron oxide nanoparticle suspensions (1 mg/ml, 10-50 nm) in water with 465 kHz RF magnetic field.
  • B TEM of Ocean Nanotech (SHP-20-50) iron oxide nanoparticles and their size distribution, calculated to be 19.83 ⁇ 2.7 from 450 particles.
  • C X-ray photoelectron spectroscopy of iron oxide nanoparticle samples. Survey indicated the presence of iron, carbon, and oxygen with iron content investigated in upper inset and the presence of carboxyl groups confirmed in the lower inset.
  • FIGURE 3 Nanoparticle decoration of cells in vitro.
  • A Nanoparticle decoration of cells in vitro. Significant nanoparticle binding to the surface of HEK293T cells expressing His tagged TRPV1 compared to untransfected cells.
  • B Electron micrograph of anti-His antibody coated iron oxide nanoparticles (20nm) binding to untransfected HEK293T cells (left panel) and HEK293 cells transfected with TRPVl His (right panel). Scale bar 200nm.
  • C Immunoelectron micrography of anti-His antibody coated iron oxided nanoparticles (20nm) co-localized with silver enhanced gold immunostaining for TRPVl (lOnm particles) in transfected HEK 293T cells (left panel). There is no TRPVl immunostaining in the absence of the primary antibody (right panel). Scale bar as indicated.
  • D Representative changes in Fluo-4 fluorescence after application of TRP agonist 2APB or RF treatment in HEK 293T cells transfected with TRPVl Hls and decorated with nanoparticles.
  • FIGURE 4 Temperature dependent release of proinsulin. Proinsulin release from HEK 293T cells transfected with calcium dependent insulin alone, TRPVl Hls and calcium dependent insulin or TRPVl and calcium dependent insulin was examined at 32°C, below the threshold for TRPVl activation and at 44°C, above the threshold for TRPVl activation. Expression of TRPVl Hls and TRPVl significantly increased proinsulin release at 44°C compared to that from cells without TRPVl . There is no significant difference in the proinsulin release seen with TRPVl Hls compared to unmodified TRPVl . (Same letter indicates p ⁇ 0.05).
  • FIGURE 5 Bioengineered human insulin construct. Calcium dependent insulin release is via three calcium response elements: serum response element (SRE), cyclic AMP response element (CRE) and nuclear factor of activated T-cell response element (NFAT RE) and a minimal promoter upstream of a furin sensitive human insulin cDNA.
  • SRE serum response element
  • CRE cyclic AMP response element
  • NFAT RE nuclear factor of activated T-cell response element
  • RF treatment does not change proinsulin release from cells expressing the calcium dependent insulin gene from cells with TRPVl Hls , calcium dependent human insulin and nanoparticles in the absence of RF treatment, from cells expressing TRPVl and calcium dependent human insulin treated with RF but in the absence of nanoparticles, or from cells treated with RF expressing calcium dependent human insulin and binding nanoparticles via a nanoparticle tether comprised of a platelet derived growth factor receptor transmembrane domain with fused extracellular biotin acceptor [protein but in the absence of TRPVl .
  • C The effects of nanoparticle heating are cell specific.
  • Proinsulin release from RF treated HEK cells transfected with TRPVl Hl 7calcium dependent human insulin incubated with anti- His oxide nanoparticles was blocked by preincubation with Tacrolimus (lOOnM). There is no difference apoptotic cells incubated with increasing consentrations of nanoparticles as assessed by TUNEL count (F) are activated Caspase-3 count (G) between untreated and RF treated cells is transfected with TRPVl His .
  • FIGURE 6 (A) RF treatment increases proinsulin release and insulin gene expression in vitro. Nanoparticle-decorated HEK293T cells transfected with TRPVl Hls and calcium-dependent insulin show a significant increase in proinsulin release and insulin gene expression with RF treatment that is blocked by the TRP antagonist ruthenium red.
  • FIGURE 7 Expression of constructs and RF dependent proinsulin release from ES cells.
  • A Expression of insulin in ES cell clones: Quantitative PCR measured expression of human insulin in 3 ES cell clones (4,6 and 7) electroporated with TRPVl Hls and Ca 2+ - dependent human insulin construct along with cells stably expressing TRPV1 alone, Ca 2+ - dependent human insulin construct alone or wild-type ES cells.
  • TRPV1 in ES cell clones Quantitative PCR measured expression of TRPV1 in 3 ES cell clones (4, 6 and 7) electroporated with TRPVl Hls and Ca 2+ -dependent human insulin construct along with cells stably expressing TRPV1 alone, Ca 2+ -dependent human insulin construct alone or wild- type ES cells.
  • C Immunohistochemistry for TRPV1 (upper panels) or His (lower panels) in wild-type cells (left panels), cells stably expressing TRPVl Hls (middle panels) or ES clone 7 (right panels).
  • D RF dependent pro insulin release from ES cells.
  • FIGURE 8. depicts dual component system for cell activation.
  • A Schema of dual component system. Streptavidin coated iron oxide nanoparticles bind biotin on a cell surface biotin acceptor protein fused to a transmembrane domain (BAPTM). Exposure to an RF field induces local heating, which opens TRPVl channels. Calcium entry triggers downstream processes as before.
  • (B) Nanoparticle binding to the surface of HEK 293 T cells expressing TRPVl and BAPTM is increased compared to untransfected cells (p 0.09).
  • FIGURE 9 In vitro and in vivo studies on PC 12 TRPVl Hi 7insulin stable cell line.
  • A Proinsulin release from PC 12 cells stably expressing TRPVl Hls and calcium dependent human insulin was significantly increased by a temperature above the threshold for TRPVl activation (Same letter indicates p ⁇ 0.05).
  • B RF treatment significantly increased proinsulin release from PC 12 cells stably expressing TRPVl Hls and calcium dependent human insulin. (Same letter indicates p ⁇ 0.01).
  • C RF treatment significantly increases insulin gene expression in PC 12 cells stably expressing TRPVl Hls and calcium dependent human insulin (Same letter indicates p ⁇ 0.05).
  • FIGURE 10 Nanoparticle regulation of blood glucose in vivo.
  • FIG. 11 Nanoparticle regulation of blood glucose in vivo.
  • A Protocol for assessment of effects of RF treatment on blood glucose in mice bearing tumors expressing TRPVl Hls and calcium dependent human insulin. At time -30 min, mice are anesthetized and injected with PBS or nanoparticles. RF stimulation begins at time 0 and continues for 30 mins. Mice remain anesthetized for a further 30 mins. Samples for plasma insulin are taken at -30 and +30 mins and samples for blood glucose are taken before, during and after RF stimulation.
  • B Expression of c-fos gene in tumors showed no difference in levels between control (untreated) and RF treated tumors.
  • C No increase in apoptotic cells from
  • nanoparticle treated mice with tumors expressing TRPVl Hls and calcium dependent human insulin.
  • RF treatment significant reduces blood glucose in nanoparticle treated mice compared to PBS treated mice in both the first and second study separated by a week.
  • FIGURE 12 Effect of NICE in the absence of TRPV1, absence of anti-His antibodies and temperature studies in vivo.
  • A Effect of RF stimulation on blood glucose in PBS and nanoparticle treated mice bearing tumors expressing calcium dependent insulin gene without TRPVl .
  • B Effect of RF stimulation on blood glucose in mice treated with PBS or nanoparticles which have not been conjugated to anti-His antibody.
  • C Thermal imaging using an infrared camera on mouse with tumor expressing TRPVl His and calcium dependent human insulin injected with iron oxide nanoparticles before (left panel) and after (right panel) RF magnetic field treatment.
  • FIGURE 13 Intracellular nanoparticle synthesis and cell activation.
  • a ferritin fusion protein is composed of a ferritin light chain fused to ferritin heavy chain with a flexible linker region. Heating of the iron core by a RF magnetic field opens the TRPVl channel to trigger calcium entry, as previously described.
  • B RF treatment increases proinsulin release in vitro.
  • FIGURE 14 Expression of Ferritin fusion protein in vitro.
  • A Ferritin expression as shown by IHC for ferritin light chain.
  • B Electron micrograph of iron loaded ferritin in transfected cells. Scale bar 200nm.
  • FIGURE 15 Release of proinsulin with RF (465kHz) from 293 cells transfected with TRPVl and myristoylated ferritin fusion protein (mFerritin), transfected with TRPVl with n-terminal fusion of came lid antibody to EGFP (vhh-TRPVl) and EGFP fused to ferritin fusion protein and transfected with TRPVl with n-terminal fusion of camelid antibody to EGFP (vhh-TRPVl), EGFP fused to ferritin fusion protein and camelid antibody fused to ferritin fusion protein along with calcium dependent insulin gene.
  • mFerritin myristoylated ferritin fusion protein
  • FIGURE 16 Release of proinsulin from (A) embryonic stem cells from C57BL6 mice expressing TRPVl, myristoylated ferritin fusion protein and calcium dependent insulin, decorated with nanoparticles and treated with RF and (B) mesenchymal stem cells from C5BL6 mice expressing TRPVl, myristoylated ferritin fusion protein and calcium dependent insulin and treated with RF.
  • FIGURE 17 Regulation of blood glucose in wild-type mice.
  • C57BL6 mice received in injection of replication deficient adenovirus expressing TRPVl, myristoylated ferritin fusion protein (mferritin) and calcium dependent insulin or adenovirus expressing LacZ. Two weeks after injection, mice were fasted overnight and anesthetized then treated with RF for 1 hour and blood glucose monitored.
  • RF treatment of TRPVl /mferritin/calcium dependent insulin significantly reduced blood glucose compared to baseline and compared to either RF treated LacZ expressing mice or mice expressing TRPVl /mferritin/calcium dependent insulin without RF treatment.
  • FIGURE 18 (A) Change in blood glucose and (B) change in blood glucose expressed as area under curve in nude mice injected with mesenchymal stem cells alone (control) and treated with RF or mesenchymal stem cells expressing TRPVl, mferritin and calcium dependent insulin and treated with RF.
  • FIGURE 19 A synthetic promoter comprised of three calcium response elements: serum response element (SRE), cyclic AMP response element (CRE) and nuclear factor of activated T-cell response element (NFAT RE) and a minimal promoter were cloned upstream of a modified, furin sensitive insulin cDNA .
  • SRE serum response element
  • CRE cyclic AMP response element
  • NFAT RE nuclear factor of activated T-cell response element
  • a minimal promoter were cloned upstream of a modified, furin sensitive insulin cDNA .
  • HEK 293t cells expressing calcium dependent human insulin and either TRPVlHis or TRPV1 BAP were decorated with functionalized 10 nanoparticles. Applying a RF magnetic field to nanoparticle-decorated cells expressing TRPVlHis or TRPVl BAP and calcium regulated furin sensitive insulin significantly increased proinsulin release and insulin gene expression.
  • FIGURE 20 shows the effect of NICE in vivo.
  • A Protocol to examine the effect of RF on blood glucose and insulin in vehicle or nanoparticle injected TRPVl /NFAT -insulin tumors in nude mice.
  • B Effect of RF on blood glucose in vehicle (PBS) or nanoparticle injected mice. A significant difference in blood glucose is seen at 30, 45 and 60 minutes.
  • C Assessment of area under the curve for circulating blood glucose shows a significant difference between PBS and nanoparticle treated groups between 0 and 120 minutes.
  • D Circulating insulin levels increase significantly in nanoparticle treated mice.
  • E Insulin gene expression, as assessed by qPCR, is significantly increased in RF treated tumors.
  • FIGURE 21 shows a schema of intracellular nanoparticle synthesis using ferritin chimeras.
  • A Iron binding chimeric ferritin peptides composed of ferritin light chain (FLC) and ferritin heavy chain (FHC) with a flexible linker sequence (pink box) are fused to either EGFP (green box) or the high affinity camelid anti-GFP antibody (yellow box). When these are expressed they form ferritin complexes with either EGFP or nanobody at the surface.
  • the nanobody peptide is also fused to the intracellular C terminal of the temperature sensitive calcium channel, TRPVl .
  • FIGURE 22 shows heating of iron oxide nanoparticles in 465kHz radiofrequency field.
  • A Exposure of 20nm ferrous oxide nanoparticle suspension (1 mg/ml) and water to 465kHz radiofrequency field
  • B Significant increase in temperature of nanoparticles (compared to water). Nanoparticle temperature increases by 5°C in 30s without any increase in water temperature.
  • FIGURE 23 shows confirmation of co-expression of TRPVl and nanoparticle tether in vitro.
  • A Dual staining for TRPVl and HA (upper panels). TRPVl and biotin (middle panels) and TRPVl and streptavidin Alexa 594 (lower panels) in transfected HEK 293t cells.
  • B Streptavidin coated iron oxide nanoparticle binding (lOnm) to transfected HEK 293t cells (left) and quantification in non-transfected and transfected cells (right).
  • FIGURE 24 shows opening of TRPVl channels and calcium entry in response to nanoparticle heating by RF in vitro.
  • A TRPVl opening and rapid calcium entry in
  • HEK293t cells transfected with TRPVl and BAPTM and decorated with streptavidin coated nanoparticles in response to nanoparticle heating in RF field. Calcium entry was measured as a change in fluorescence intensity of the calcium indicator Fluo-4.
  • B Pseudocolored images indicating change in fluorescence intensity in TRPVl transfected cells with RF stimulation.
  • C Indirect assessment of intracellular calcium via expression of luciferase under the control of a calcium dependent NFAT promoter.
  • Luciferase expression is significantly increased in HEK293t cells only in the presence of all components of the NICE system: TRPVl and the biotin acceptor protein (BAP), the addition of 20nm iron oxide nanoparticles (NP) and the presence of 465kHz, 1 lOkA/m electromagnetic field (RF).
  • FIGURE 25 shows constructs for viral delivery of NICE components and RF dependent hormone release.
  • A Construct for constitutive expression of NICE components, BAPTM and TRPVl .
  • B Construct for calcium dependent expression of furin modified human insulin.
  • C Construct inserted into adenovirus for ere dependent expression of NICE components, BAPTM and TRPVl using FLEX system.
  • D 293t cells transiently trans fected with TRPVl -Baptm and NFAT insulin show a significant increase in proinsulin release with nanoparticle binding and RF exposure.
  • E Insulin gene expression is also significantly increased in these cells with RF exposure.
  • FIGURE 26 shows combinatorial activation of transfected cells.
  • a mixture of 2 cell populations will be studied each expressing a unique linear epitope in the first extracellular loop of TRPVl to tether an antibody coated nanoparticle tuned to a distinct wavelength. Subsequent calcium entry increase expression of a calcium dependent luciferase unique to each cell population.
  • Iron oxide nanoparticles coated with anti-His 6X antibody bind to His 6 epitope in TRPVl . Calcium entry triggers CBR luc expression.
  • B Gold nanoparticles coated with anti-FLAG antibody bind to FLAG epitope in TRPVl . Calcium entry triggers CBG99 luc expression.
  • FIGURE 27 shows constructs for generation of transgenic mice for expression of NICE components.
  • Transgenic mice will be generated with the insulin promoter driving expression of TRPVl (A) and BAPTM (B). These mice are crossed to express both TRPVl and BAPTM in beta cells (C).
  • An additional transgenic mouse with luciferase downstream of NFAT response elements will act as an in vivo reporter of intracellular calcium (D).
  • the resulting mice (E) will express TRPVl and BAPTM in beta cells and calcium dependent luciferase in all cells.
  • FIGURE 28 shows an illustrative scheme for self-stimulation protocol with lickometer:
  • A a fiber connector for implant of a biocompatible 200 ⁇ fiber optic;
  • B implanted fiber to deliver light to the ventral tegmental area;
  • C Med associates photobeam lickometer;
  • D the self-stimo-lick paradigm is a variation of the self-stimulation paradigm where the operant behavior is a lick.
  • Light stimulation of ChR2 positive neurons or RF stimulation of NICE positive neurons occurs only when the mouse consumes water from the lickometer;
  • E animals injected with AAV-Flex-hChR2-mCherry virus (top) consume more water than controls (bottom) form the connected port.
  • F cumulative licks over a 2 hour trial Light sensitive (red) and control animals (blue) in a SSL paradigm.
  • FIGURE 29 shows expression of NICE constructs in Agrp and POMC neurons.
  • A generation of BAC transgenic mouse with His tagged TRPV1 under the control of Agrp promoter
  • B transgenic mouse with Flag-tagged TRPV1 under control of POMC promoter
  • FIGURE 30 Iron oxide (IO) nanoparticles functionalized with monoclonal antibodies against the His x 6 epitope tag are targeted to cells.
  • IO Iron oxide
  • A-B In the presence of a RF field, local heating of IO nanoparticles above the threshold for TRPV1 channel activation (42°C) triggers calcium entry and cell activation.
  • FIGURE 31 Depiction of a modified TRPV1 channel as both a nanoparticle tether and effector.
  • FIGURE 32 No significant increase in proinsulin release was observed in HEK 293t cells decorated with IO nanoparticles in the presence of an RF magnetic field without TRPV1 (transfected with BAP only), in cells with TRPV1 but without nanoparticle binding (transfected with TRPV1 only), or in cells with nanoparticles and TRPV1 (TRPVl Hls or TRPV1 BAP) but in the absence of the RF magnetic field.
  • FIGURE 33 In vitro studies examining the effects of RF treatment on proinsulin release and insulin gene expression replicated the findings in transfected HEK 239t cells (A - C).
  • Stably transfected PC12-TRPVlHis-Ins cells were injected subcutaneously into the flank of nude mice and formed tumors expressing TRPVlHis (D) and furin sensitive insulin constructs. Following an overnight fast, PBS or 10 nanoparticles were injected into the tumors of anesthetized mice (50ul total volume, nanoparticle concentration 8mg/ml). Blood glucose and plasma insulin were measured before, during and after the application of an RF field (E).
  • the present invention provides methods and compositions for the remote control of cell function based on the use of radiofrequency waves to excite nanoparticles targeted to specific cell types.
  • the cell type of interest expresses a temperature sensitive channel wherein excitation of the nanoparticles results in a localized temperature increase than is transduced into a cellular response.
  • Such cellular responses include for example, modulation of cell proliferation, cell differentiation, apoptosis, and/or activation of signal transduction pathways.
  • the cellular response is an increase in gene expression resulting in production of one or more physiologically active proteins.
  • proteins may be used to treat various inherited or acquired disorders including for example, cardiovascular disorders, central nervous system disorders, autoimmune diseases, oncological diseases, hormonal disorders, metabolic diseases, blood disorders or immune disorders. Additionally, the proteins may be expressed to treat various infectious diseases including, for example, viral, bacterial, parasitic, and fungal infections.
  • the cellular response resulting from nanoparticle excitation may also be designed to result in an increase in gene expression resulting in production of one or more nucleic acid molecules of interest.
  • nucleic acid molecules include those molecules capable of regulating protein expression, such as antisense and siRNA molecules.
  • the expression system of the present invention can be used with virtually any type of biological cell population, including prokaryotic and eukaryotic cells.
  • eukaryotic cells include, for example, plant and mammalian cells.
  • the specific cell type used will typically vary depending upon the type of cellular response that is sought to be regulated.
  • mammalian cells and specifically, human cells or animal cells are typically preferred for increased expression of a physiological protein for use as a therapeutic.
  • the cell type of interest is a stem cell, preferably a mammalian stem cell.
  • stem cells engineered to express NICE constructs can act as autografts to enable external control of cell function.
  • stem cell refers to any cell having the potential to differentiate into one or more different cell types, including pluripotent stem cells.
  • Such cells include, but are not limited to, stem cells derived from a variety of different sources including, for example, bone marrow, embryonic blastocysts or yolk sac, spleen, blood, including peripheral blood and umbilical cord blood, adipose tissue and other tissues and organs.
  • stem cells include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells, endothelial progenitor cells or embryonic stem cells.
  • the cell type of interest expresses a temperature sensitive channel wherein activation of the nanoparticles results in a localized temperature increase that is transduced into a cellular response via the temperature sensitive channel.
  • temperature sensitive channels include, but are not limited to, the TRPV1 channel TRPV2, TRPV3, TRPM8, chimeric TRP channels, TREK-2 and tandem pore domain potassium channels, such as TREKl, TREK2, and TASK.
  • the localized temperature increase mediated by the excitation of the nanoparticles leads to an activation of the TRP VI channel resulting in gating of Ca2+ entry.
  • the cell type of interest expresses a channel wherein activation of the nanoparticle results in motion of the nanoparticle than is transduced into a cellular response via the mechanical motion of the particle.
  • Such motion sensitive channels include, but are not limited to, the TREK-1, TRAAK, TRPV4, TRPV1.
  • the localized stimulation of nanoparticle motion leads to an activation of the channel resulting in modulation of cell activity.
  • the cells to be targeted may be genetically engineered to express one or more genes encoding physiologically active proteins of interest, such as those proteins providing a therapeutic benefit.
  • the cells are genetically engineered in such a way that expression of the protein(s) of interest is induced in the cell upon excitation of the nanoparticles which results in a localized temperature increase or an increase in nanoparticle motion.
  • the cells to be targeted may be engineered to express a non-encoding nucleic acid molecule of interest such as an antisense or siRNA molecule.
  • the target cells maybe genetically engineered to express a temperature sensitive protein, such as a temperature sensitive ion channel, wherein an increase in temperature mediated by the excited nanoparticles results in a cellular response through activation of the ion channel.
  • target cells may be engineered to intracellularly express a protein that is capable of acting as an activated nanoparticle upon exposure to a RF magnetic field.
  • proteins include for example, the iron storage protein ferretin.
  • proteins may be expressed in the cell as fusion proteins to target their location to a specific site within the cell, for example, in close proximity to a temperature sensitive channel.
  • recombinant expression vectors designed to express a physiologically active protein of interest, or a nucleic acid molecule of interest, such as antisense or RNAi molecules, or a protein that may act as a natural nanoparticle can be introduced into the target cells of choice.
  • the recombinant expression vectors in addition to containing a nucleic acid encoding the protein or nucleic acid of interest, will contain transcriptional regulatory sequences that can be induced upon excitation of the particles resulting in expression of the protein, or nucleic acid molecule of interest.
  • transcriptional regulatory sequences include, but are not limited to, promoter and/or enhancer sequences that induce gene expression in response to ion channel activation.
  • regulatory sequences include, but are not limited to the calcium response elements, referred to herein as SRE, CRE and NFAT RE.
  • the cells may be genetically engineered using techniques well known in the art.
  • Such techniques include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook J et al. 2000. Molecular Cloning: A Laboratory Manual (Third Edition), and Ausubel et al (1996) Current Protocols in Molecular Biology John Wiley and Sons Inc., USA). Any of the methods available in the art for gene delivery into a host cell can be used according to the present invention to deliver genes into the target cell population. Such methods include electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • a viral vector that contains a nucleic acid encoding the protein or nucleic acid of interest and a transcriptional regulatory sequence that can be induced upon excitation of the nanoparticles can be used.
  • viral vectors include for example, retroviral, adenoviral or adeno-associated viral vectors. (See, Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 for a review of adenovirus- based gene delivery).
  • RF radiofrequency
  • RF signals at low and medium frequencies penetrate tissues freely and without significant energy absorption making it now possible to adapt this system for in vivo use (Jokela International Union of Radio Science 2008).
  • metallic/metal oxide nanoparticles placed in an alternating RF field absorb energy and heat in a controlled manner depending on the strength of the field, a process known as induction heating (Fortin et al., J. Am, Chem. Soc. 129:2628-2635).
  • the heating capacity depends on nanoparticle composition, size, perhaps shape and the frequency and power of the RF field and, as such, it is possible to regulate the heat generated within the physiological temperature range.
  • nanoparticles employed, for example, magnetic iron oxide and gold spheres are easily prepared, have little or no intrinsic cell toxicity and can readily be adapted to target cells by incorporating streptavidin, antibodies, or pharmacological agents (Samanta B. et al. J Mater Chem 18: 1204-1208; Wang AZ. et al. 2008 Expert Opin Biol Ther 8: 1063-1070).
  • Nanoparticles of differing compositions and shapes are heated at defined rates by different electromagnetic field frequencies and strengths.
  • Nanoparticles for use in the present invention include, but are not limited to, metallic nanoparticles, and metal oxide
  • nanoparticles include, but are not limited to, iron oxide nanoparticles, gold nanoparticles, and the like.
  • iron oxide nanoparticles are maximally heated by an EM frequency of around 465kHz while gold nanoparticles heat at an EM frequency of 13.5MHz, with the field strength determining the rate of heating.
  • This property potentially allows distinct EM frequencies to differentially heat nanoparticles of different compositions and/or shapes.
  • Nanoparticles consisting of different chemistries, such as, but not limited to, gold and iron oxide, and of different shapes, particularly nanoparticles of different aspect ratios (e.g., spheres vs. rods), can be chosen based on their discrete heating frequencies and resistivities.
  • the described invention provides non- limiting, illustrative compositions and methods encompassing different chemistries and spheres of specific sizes.
  • the nanoparticles can be directed to distinct cell populations via cell-specific expression of unique tethers and then, using RF generators and tunable amplifiers with a range of excitation frequencies, excite different cell populations alone or in combination.
  • the nanoparticles can be conjugated to various biological or chemical moieties that bind a specific receptor or target a specific cell type.
  • the nanoparticles may be externally applied to the cells.
  • the ligand can comprise a small molecule, peptide, antibody, nucleic acid, protein, carbohydrate, lipid, polyethylene glycol derivatives or any combination thereof.
  • Metal nanoparticles can readily be functionalized to target define cell populations by coating with specific antibodies that recognize proteins that are normally expressed on a cell or transfected into that cell (Samanta et al., J. Nat. Chem. 18: 1204-1208; Wang et al, Expert. Opin. Biol. Ther. 8: 1063-1070).
  • streptavidin which binds to biotin with extremely high affinity
  • a system is provided whereby streptavidin-conjugated nanoparticles can be coupled to biotin-labeled cells through the strepavidin/biotin high-affinity reaction. Loading of nanoparticles onto the cells permits targeting of heat to said cells.
  • cells may be genetically engineered to express proteins which can act as naturally occurring nanoparticles and which can be activated by a RF magnetic field.
  • proteins include, for example, the iron storage protein ferritin, the bacterial gene MagA, ceruloplasmin and transferrin.
  • the method of the present invention comprises contacting a target cell population with nanoparticles for a time sufficient to permit binding of the nanoparticle to the surface of the target cell.
  • the nanoparticles are administered in vivo to a subject resulting in contacting of a target cell population with nanoparticles for a time sufficient to permit binding of the nanoparticle to the surface of the target cell.
  • the target cells are cultured, using routine tissue culture methods well known to those of skill in the art. Cells are then washed with a buffer, such as a phosphate-buffered saline (PBS) and a solution of nanoparticles is added to the target cells.
  • PBS phosphate-buffered saline
  • the nanoparticle solution comprises a mixture of the tissue culture media in which the cells are cultured and nanoparticles. Cells are incubated with the nanoparticles for a time sufficient to permit efficient binding of the nanoparticles to the target cells. Transfer of nanoparticles to the target cells can be monitored using, for example, flow cytometry.
  • the target cell population are stem cells. Also within the scope of the invention are nanoparticle labeled cells that have been genetically engineered to express a desired protein, or nucleic acid of interest. For example, nanoparticle labeled cells may be engineered to express proteins capable of providing a therapeutic benefit.
  • the present invention further provides pharmaceutical compositions comprising nanopartices, nanoparticle-labeled cells and/or cells that express nanoparticles intracellularly and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer, phosphate-buffered saline (PBS), or 0.9% saline.
  • PBS phosphate-buffered saline
  • Such carriers also include aqueous or non-aqueous solutions, suspensions, and emulsions.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Preservatives and other additives such as, for example, antimicrobials, antioxidants and chelating agents may also be included with all the above carriers.
  • Nanoparticle-labeled cells and/or cells that express nanoparticles intracellularly can also be incorporated or embedded within scaffolds which are recipient-compatible and which degrade into products which are not harmful to the recipient. These scaffolds provide support and protection for nanoparticle-labeled cells that are to be transplanted into the recipient subjects.
  • the present invention provides methods and compositions which may be used to provide a therapeutic benefit for treatment of various diseases. Specifically, through the use of nanoparticle-labeled cells and/or cells that express nanoparticles intracellularly, a therapeutic protein, or nucleic acid molecule of interest, may be delivered to a subject in need of treatment through administration of nanoparticle labeled cells. Alternatively,
  • nanoparticles themselves may be administered to a subject in need of treatment, wherein said nanoparticles are targeted to endogenous cells of the subject wherein excitation of the nanoparticle results in a localized temperature increase that is transduced into a cellular response.
  • compositions may be formulated in any conventional manner using one or more physiologically acceptable carriers optionally comprising excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen.
  • the methods of the invention comprise administration of nanoparticles and/or nanoparticle labeled cells and/or cells that intracellularly express a nanoparticle, in a pharmaceutically acceptable carrier, for treatment of various disorders or diseases.
  • administering shall mean delivering in a manner which is effected or performed using any of the various methods and delivery systems known to those skilled in the art. Administering can be performed, for example, pericardially, intracardially, subepicardially,
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carvers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
  • compositions will contain a therapeutically effective amount of the therapeutic compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the therapeutic compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions of the invention which will be effective in the treatment of a particular disorder or disease will depend on the nature of the disorder or disease, and can be determined by one of skill in the art using standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose response curves derived from in vitro or animal model test systems. Additionally, the administration of the compound could be combined with other known efficacious drugs if the in vitro and in vivo studies indicate a synergistic or additive therapeutic effect when administered in combination.
  • the progress of the recipient receiving the treatment may be determined using assays that are designed to detect the physiologically active protein expressed by the nanoparticle targeted cells.
  • the present invention further relates to transgenic non-human animals that may be engineered to contain cells that respond to nanoparticle excitation in a desired fashion.
  • the transgenic animals may be engineered to express cell surface receptors that act as binding partners for the nanoparticles.
  • Said target cells may either naturally, or through genetic engineering, express a protein, or nucleic acid molecule of interest upon nanoparticle excitation.
  • the transgenic animals may be engineered to intracellaulary express a nanoparticle, such as for example a naturally occurring iron nanoparticle.
  • Such transgenic animals provide in vivo model systems for studying normal physiological processes as well as disease processes.
  • the transgenic animals of the invention may further be useful as in vivo model systems for use in identification and testing of novel therapeutic compounds of interest.
  • the method of the invention can be implemented as follows. First, expression of both a biotin acceptor protein (BAP) fused to the transmembrane domain of platelet derived growth factor receptor (PDGFR) as a tether for streptavidin coated nanoparticles, and TRPV1, a temperature sensitive cation channel can be expressed in specific cells using specific promoters. Second, cells are "decorated "by delivering streptavidin-coated iron oxide nanoparticles into the region where the BAP is expressed. The high affinity of streptavidin and biotin results in the cells being coated with the metallic/metal oxide nanoparticles. Studies in vitro have confirmed this to be the case. Third, the cultured cells, or an animal, are exposed to a RF field of defined strength at an intensity that will increase the local temperature of the nanoparticle decorated cells, activate the TRPV1 channel and thus triggering calcium influx.
  • BAP biotin acceptor protein
  • PDGFR platelet derived growth factor receptor
  • the system can be modified such that only one construct is used for both tethering of the nanoparticle and gating of Ca2+ entry.
  • the TRPVl protein may be engineered as a fusion protein capable of direct tethering of the nanoparticle.
  • the TRPVl protein may be engineered as a fusion protein containing any "tag" that does not interfere with the functioning of the channel.
  • tags are well know to those of skill in the art.
  • the TRPVl protein can be expressed as a fusion protein containing HIS tags. In such a case, the nanoparticles are coated with anti-HIS antibodies for targeting to the cell.
  • the present invention provides methods and compositions for studying the role of different cell types in a complex organism.
  • the definitive test of cell function is to selectively turn on or off the activity of a single cell type in a living animal and examine the effect on physiological function.
  • the present invention provides for the use of nanoparticles to activate defined cell populations remotely with radiowaves.
  • ferrous oxide coated with streptavidin can be used to decorate cells, which express a biotin acceptor protein under the control of cell specific promoters.
  • These same cells are engineered to also express TRPV1, a single component, temperature-sensitive ion channel that can detect small changes in temperature within the physiological range and by conformational change allow graded calcium entry.
  • Exposing the metal coated cells to a defined electromagnetic field increases the local temperature and activates TRPV1 channels resulting in a Ca2+ current and cell activation.
  • Data is provided below that confirms the efficacy of this method both in vivo and in vitro.
  • the technology can be used to modulate functions such as hormone release and neural activity.
  • a means is also provided for combinatorial activation of different cells using a modified TRPV1 and nanoparticles fabricated from other metals that can be excited at different wavelengths. This tool can be used, for example, to examine the roles of specific peripheral and CNS cell populations in energy metabolism.
  • the methods and compositions of the invention can be used to refine the methodology by decorating different cell types with distinct particles tuned to different wavelengths to activate ensembles of different cell populations in various combinations. Further, the ability of NICE to modify hormone release to regulate glucose metabolism in diabetic animals in vivo can be further refined.
  • the methods and compositions can also be used for stimulation of action potentials in electrically excitable cells to modify behavior and can be used to study the role of specific hypothalamic populations in (NPY and POMC) control of appetite.
  • the present invention provides methods and compositions to remotely and selectively switch on the activity of a single cell in a living organism and examine the effects on physiological function using nanoparticle induced cell excitation (NICE).
  • NICE nanoparticle induced cell excitation
  • the technique targets temperature sensitive calcium channels (TRPV1) to defined cells. These cells are decorated with metal nanoparticles which are heated by an external radiofrequency field. This in turn opens the TRP channel to stimulate calcium influx. Calcium entry initiates downstream events such as depolarization (neurons), hormone release (endocrine cells) or gene expression.
  • TRPV1 temperature sensitive calcium channels
  • the studies described herein demonstrate the efficacy of NICE at stimulating calcium influx, modulating hormone release and stimulating gene expression both in vivo and vitro.
  • Nanoparticles Iron oxide nanoparticles (10-50 nm diameter), functionalized with a surface carboxylic acid group, were purchased from Ocean Nanotech (Springdale, Arkansas). The nanoparticles were conjugated to either mouse monoclonal anti-His antibody (AbD Serotec, Raleigh, NC) or streptavidin (Jackson Immunoresearch).
  • RF magnetic-field A 465 kHz sinusoidal signal was provided by a signal generator and applied through an amplifier (both Ultraflex, Ronkonkoma, NY) to a 2-turn solenoid coil with a radius of 2.5 cm to produce a magnetic field strength of 5 mT. Samples were placed within the solenoid. A 13.56 Mhz sinusoidal signal was provided by a signal generator (RF Instrumentation, PA) and applied through an amplifier (Comdel, Gloucester, MA) to a 2-tum solenoid coil with a radius of 2.5 cm).
  • Plasmids. TRPV1 On pcDNA3.1 was a kind gift of Wolfgang Liedkte (Duke University, NC) and cloned into pEGFP-Nl (Clontech, Mountainview, CA). It was modified by PCR (Fast start PCR, Roche) to introduce Hisx6. Nuclear factor of activated t- cells (NFAT) response elements and minimal promoter were from pGL4.30[/wc2P/NFAT- RE/Hygro (Promega, Madison, WI). Serum response element (SRE), cyclic AMP response element (CRE) and form modified human insulin sequences were synthesized by Integrated DNA technologies (Coralville, IO).
  • BAPTM the transmembrane domain of platelet derived growth factor receptor fused to an extracellular biotin acceptor protein, was a kind gift of Dr. B Tannous, Massachusetts General Hospital, MA).
  • TRPVl His and calcium responsive form insulin were cloned into MSCV-hygro and MSCV- puro plasmids (Clontech,) respectively for retrovirus production using Phoenix packaging cells.
  • Mouse ferritin heavy chain was obtained from ATTC (Manassas, VA) in pCMV sport6 and mouse ferritin light chain 1 was obtained from Invitrogen (Carlsbad, CA) in pYX-Asc. These were cloned downstream of EF1 alpha promoter in pCR2.1 with a flexible linker region to create ferritin light chain— linker— heavy chain fusion protein. The fidelity of PCR products was confirmed by DNA sequencing.
  • HEK 293T Human embryonic kidney cells
  • PC12 cells were cultured in RPMI medium 1640 with 10%) horse serum and 5% fetal bovine serum (Gibco) at 37°C and 5% C0 2
  • Phoenix ecotropic packaging cells (Stanford University) were grown in Dulbecco's modified eagle medium with 10%> fetal bovine serum (Gibco) at 37°C and 5% C0 2 .
  • Stable cell lines were produced by retroviral infection of PC 12 cells using the Phoenix system. Briefly, Phoenix eco cells (2 x 10 6 cells per 6-cm dish) were transfected with MSCV-puro or hygro plasmids as described above. After 24 hours, the medium was replaced and the cells placed at 32°C. Medium was aspirated after a further 24 h and spun to remove cell debris. The Phoenix cell supernatant was added to PC 12 cells (plated at 1 x 10 6 cells per 6-cm dish) using a 1 :2 dilution in RPMI medium/10%FBS with polybrene(4 ⁇ / ⁇ 1, Sigma-Aldrich, St Louis, MO). Cells were incubated at 32°C for a further 24 h before replacing the medium with RPMI/10% FBS. Selection medium was added 48 h after infection.
  • Embryonic stem cells were electroporated with a calcium dependent human insulin plasmid and selected with puromycin for 3 weeks. Resistant cells were identified for insulin insertion by Southern blot analysis before electroporation with TRPVlHis plasmid and selection with hygromycin for 3 weeks. Three double resistant clones were screen by quantitative PCR for expression of insulin and TRPVl and by
  • RF dependent release of calcium dependent human insulin 24 h prior to the study, cells were placed in serum free medium at 32°C to ensure minimal activation of TRPVl and calcium dependent pathways. On the day of study, cells were preincubated for 30 min in 500 ⁇ 1 PBS or 500 ⁇ of functionalized iron oxide nanoparticles (1 mg/ml) resuspended in PBS. Cells were washed three times in PBS before incubation in 300 ⁇ of calcium imaging buffer at room temperature (control) or in a RF field at room temperature. The supernatant was removed at 15, 30, 45 or 60 min depending on the study, spun to remove cell debris and frozen at -80°C until assay. For gene expression analysis, cells from the supernatant and cover glass were lysed and the lysate stored at 80°C until RNA purification. For apoptosis studies, the cells were incubated with functionalized
  • nanoparticles at 1, 2, 4 or 8 mg/ml before RF treatment and immunocytochemistry.
  • RF dependent time course from stable PC 12 cells, cells were left in serum containing medium prior to treatment to more accurately replicate conditions in vivo before performing the studies as described above. Medium was removed for assay at 15 and 30 mins.
  • ruthenium red was added to the calcium imaging buffer.
  • Tacrolimus (lOOnM, Tocris Bioscience, Minneapolis, MN) was added to the medium for 24 hours prior to RF treatment.
  • 3-cm dishes of HEK 293T cells were trans fected with either BAPTM or TRPVl and calcium dependent human insulin. After 24 h, the two transfected cell populations were trypsinized and mixed to form a co-culture with adjacent cells expressing BAPTM and TRPVl /human insulin.
  • ICC Immunocytochemistry
  • Control or RF treated transfected cells were washed and fixed as above then incubated in for 1 h in blocking buffer (3% BSA (Sigma) and 2% goat serum (Sigma) in PBS with 0.1% Triton-X (Sigma)). Following blocking cells were incubated in primary antibody, rabbit anti-NFATl (Cell signaling), 1 :50, diluted in blocking buffer overnight at 4 degrees. Cells were then washed three times in PBS before incubation in secondary antibody (goat anti-rabbit 488 1 : 1000) diluted in blocking buffer for 1 h. The cells were then washed a further three times in PBS before inverting and mounting using Fluoromount with DAPI (Southern Biotech, Birmingham, AL).
  • ICC was also used to examine expression of ferritin fusion.
  • Cells were washed twice with PBS, fixed and blocked as above. Cells were incubated in primary antibody, rabbit anti-ferritin light chain (Dako, Carpinteria, CA), 1 : 1000, diluted in blocking buffer for 1 h. Cells were washed three times in PBS before incubation in secondary antibody (goat anti-rabbit 488 1 : 1000) diluted in blocking buffer for 1 h. The cells were washed a further three times in PBS before inverting and mounting using Fluoromount (Southern Biotech, Birmingham, AL).
  • Immunohistochemistry was used to confirm expression of TRPV1 and His and quantify apoptotic cells in tumors. Tumors were fixed in 10% formalin (Sigma) at 4°C overnight then placed in 30% sucrose in PBS at 4°C for a further 24 h. Tissue was embedded in OCT and frozen before 20 ⁇ cryosections were cut and placed directly on glass slides. Slides were placed at 55 °C for 1 h then stored at -80 °C before staining.
  • Apoptag Fluorescein direct in situ apoptosis detection and IHC for activated caspase-3 were performed according to the manufacturers' instructions. Staining for TRPV1 and His was performed as follows. Slides were washed three times with PBS then incubated in blocking buffer for 2 h followed by overnight incubation at 4°C with primary antibody diluted in blocking buffer (rabbit anti-TRPVl 1 :500, mouse anti-His 1 : 1000 (Sigma)). Slides were washed 3-times in PBS and then incubated overnight at 4°C with secondary antibody diluted in blocking buffer (goat anti-rabbit 488 and goat anti-mouse 594 both at 1 : 1000).
  • Electron microscopy was used to quantify nanoparticle binding to the cell membrane and to image ferritin in trans fected cells. Cells were fixed in 2%
  • Blocks were cut with a diamond knife on a Leica UltracutE (Buffalo Grove, IL) and ultra-thin ( ⁇ 70nm) sections were collected on uncoated 200mesh grids and stained with uranium and lead. Grids were viewed with a Tecnai SpiritBT Transmission Electron Microscope (FEI, Hillsboro, Oregon) at 80 KV and pictures were taken with Gatan 895 ULTRASCAN Digital Camera (Pleasanton, CA).
  • ImmunoEM was used to confirm binding of nanoparticles to TRPV1.
  • HEK 293T cells were seeded on Aclar (Ted Pella Inc, Redding, CA) in 24 well plates and transfected with TRPVlHis 24 hours later. 72 hours after transfection, cells were washed twice with PBS and incubated with anti-His coated iron oxide nanoparticles for 30 mins. Following two further PBS washes, cells were fixed in a fixative containing fresh 4.0%
  • NCI-Frederick Male athymic NCr-nu/nu mice (NCI-Frederick, 6 weeks old), an outbred strain, were used and housed under controlled light conditions (12 h light/12 h dark) and temperature (22°C), single-caged, and fed ad libitum on standard mouse chow. Animal care and experimental procedures were performed with the approval of the Animal Care and Use Committee of Rockefeller University (protocol 11421) under established guidelines.
  • mice are outbred and had high inter-individual variability in blood glucose, the studies were performed with a cross-over design with each mouse receiving first PBS and then one week later receiving nanoparticle injections. Nanoparticle injections could not be performed first because the particles remain within the tissue for prolonged periods (Giustini et al, 2011 Nanotechnology 22:345101).
  • mice received a second injection of anti-His conjugated nanoparticles, were treated with RF and blood glucose and insulin were measured.
  • Proinsulin was measured in cell supernatants by ELISA (Alpco, Salem, NH) according to manufacturer's protocol. Blood glucose was determined using a Breeze 2 glucometer (Bayer; Leverkusen, Germany). Blood was spun for 10 min and plasma was collected. Plasma levels of human insulin, produced by xenografts, were determined in mouse plasma by human specific ELISA (Alpco).
  • Nanoparticle Characterization Iron oxide nanoparticles purchased from Ocean Nanotech (Catalog # SHP-20-50) were further characterized. The particles were coated with oleic acid and a proprietary polymeric coating, which is carboxy-terminated for further functionalization. The particles were specified to be 20 nm in diameter with a standard deviation of ⁇ 5%. This was confirmed in the current work; TEM analysis of the size distribution yielded an average diameter of 19.8 ⁇ 2.7 nm ( Figure 3B). The particles were estimated to have a 4 nm oleic acid/polymeric coating in addition to the nanoparticle diameter observed in TEM. X-ray photoelectron spectroscopy (XPS) allowed identification of all elements within ⁇ 5 nm of the nanoparticle surface, as well as a more precise investigation of the nanoparticle surface chemistry and iron oxide content. XPS
  • Hyperthermia 25:499) and is complicated by factors such as the heat capacity of the surrounding medium, the nanoparticle ligand coating, and diffusion away from the nanoparticle surface.
  • the change in temperature in the vicinity of the nanoparticle decreases as the inverse of the radius, and may be approximated by the conductive heat transfer equation (Fourier's Law)(Rabin, 2002 Int. J. Hyperthermia 18: 194): T P Q l qpD 2
  • V np is the nanoparticle volume
  • Q is the total dissipated heat
  • r is the nanoparticle radius
  • q is the heating rate (W g "1 )
  • p is the nanoparticle density
  • D is the diameter of the heated volume
  • k is the thermal conductivity of water (0.64 W m "1 °C _1 ).
  • FIG. 1 A system has been developed that allows remote activation of protein production by engineered cells in vitro and in vivo.
  • the method (Figure. 1) uses iron oxide nanoparticles (FeNPs) that are coated with antibodies against His (anti-His) and that bind a modified TRPVl channel with an extracellular His x 6 epitope tag (TRPVl His ).As disclosed herein RF treatment, local heating of bound anti-His FeNPs activates the temperature-sensitive TRPVl, resulting in a calcium current to activate a Ca 2+ -sensitive promoter placed upstream of a modified human insulin reporter gene.
  • FeNPs iron oxide nanoparticles
  • anti-His antibodies against His
  • TRPVl His extracellular His x 6 epitope tag
  • FeNPs was used for the following reasons: They heat at 465 kHz, a relatively low frequency that minimizes tissue heating; particles of 20 nM or less diffuse in the extracellular space (Wang et al., 2008 Expert Opin. Biol. Ther. 8: 1063; Deflaco et al. 2001 Science 291 :2608; Thorne et al, 2006 Proc. Natl.Acd. Sci USA 103:5567); and these particles can be derivatized with antibodies.
  • 465 kHz (5 mT) substantial heating was observed for 20- and 25-nm FeNP suspensions ( Figure 2).
  • a 20-nm FeNP suspension had an initial heating rate of 0.15°C/s and a specific absorption rate (SAR) of 0.63 W/g, whereas the SAR of water at this field frequency and strength was less than 0.004 W/g.
  • SAR specific absorption rate
  • a His-tag insertion into the first extracellular loop of TRPVl provided a site for significant and specific FeNP binding (Figure 3) with direct heat transfer to the adjacent channel ( Figure 4).
  • Human embryonic kidney (HEK) 293T cells expressing TRPVl His and decorated with 20 nM FeNPs conjugated to anti-His showed a significant increase in intracellular Ca 2+ after 10s of RF exposure (Figure 3D).
  • HEK 293T cells expressing the Ca 2+ -dependent human insulin construct and TRPVl His were incubated with functionalized FeNPs.
  • RF treatment of the FeNP-decorated cells resulted in a significant increase in proinsulin release (RF -treated 671 ⁇ 235% (SEM) basal versus 100 ⁇ 13.9% for controls, P ⁇ 0.02) and insulin gene expression (RF-treated 2.20 ⁇ 0.53 insulin gene expression relative to basal versus 1.0 ⁇ 0.18 for controls, P ⁇ 0.05). These were blocked by the TRP channel inhibitor, ruthenium red (Figure 6A).
  • a fusion peptide of ferritin light chain, flexible linker region, and ferritin heavy chain fixes the ratio of light to heavy chains and increases iron binding (Iordanova et al., 2010 J.Biol. Inorg. Chem. 15:957).
  • Transfecting cells with ferritin fusion protein resulted in 12.6 ⁇ 2.86 ferritin particles per 0.2 ⁇ 2 with an average distance to the cell membrane of 60.3 ⁇ 2.85 nm ( Figure 14B).
  • both externally applied and endogenously synthesized nanoparticles can be heated by radio waves to remotely activate insulin gene expression and secretion.
  • RF-mediated cell activation does not require a permanent implant, and the cells to be activated can be localized (when using exogenous nanoparticles) or dispersed (by using genetically encoded nanoparticles).
  • Genetically encoded ferritin nanoparticles may also provide a continuous source of nanoparticles for cell activation.
  • an epitope- tagged channel offers the choice of activating distinct cell populations in the same organism with different RFs to selectively and independently heat nanoparticles bound to cell specific tags.
  • mutations of ferritin that alter the metal it encapsulates could enable combinatorial cell activation (Butts et al., 2008 Bichemistry 47: 12729).
  • a noninvasive, nonpharmacological means has been developed for cell stimulation and validated it in vitro and in vivo.
  • This system provides a useful tool for basic research and represents an initial step toward noninvasive regulation of protein production for possible therapeutic purposes.
  • This approach could be used to treat protein deficiencies by providing regulated expression of proteins that are difficult to synthesize or to deliver [such as central nervous system (CNS) replacement of hexosaminidase A for Tay-Sachs] or to allow CNS delivery of recombinant antibodies to treat brain metastases.
  • CNS central nervous system
  • This approach could also enable the activation of other Ca 2+ -dependent processes, such as muscle contraction or firing of action potentials.
  • the results demonstrate the usefulness of NICE as a tool for non-invasive stimulation of cell function in vivo. Additional studies can be performed to examine the effect of stimulating endogenous cell populations using transgenic mice expressing a nanoparticle tether and TRPVl, with or without calcium dependent gene expression. It is also possible to examine the effects of stimulating cell populations derived from individual- specific induced pluripotent stem cells (iPSC) injected into immune-competent mice. Viral free iPSC populations can be reprogrammed to a wide variety of tissues and also modified to express NICE components. These cell populations would allow the non-invasive study of specific stimulation of defined cell types and/or defined genes within these cells providing a valuable tool in the investigation of cell function particularly as it pertains to complex behaviors.
  • iPSC individual-specific induced pluripotent stem cells
  • NICE technology requires external nanoparticle application by incubation (in vitro) or injection (in vivo). For peripheral tissues, this may be achieved by intravenous administration but modulation of neuronal activity requires intracerebral administration. This is advantageous in some situations where stimulation of an anatomically defined subpopulation of a dispersed neural group is needed.
  • chimeric ferritin proteins targeted to an intracellularly tagged TRPVl channel is being studied.
  • the iron storage protein, ferritin is a complex of heavy and light chains that is capable of binding 4500 ferric ions or picograms of iron per cell. This iron binding capacity has allowed ferritin to be utilized as an MRI contrast agent in many studies.
  • Two chimeric ferritin peptides have been developed, one fused at the N-terminal to Egfp and one fused to a highly stable, high affinity (subnanomolar) camelid anti-gfp antibody known as a nanobody (Figure 21 A). These adaptations allow adjacent ferritin complexes to bind to form ferritin aggregates and EM images show these are of the order of 40-5 Onm diameter (Figure 2 IB).
  • the temperature dependent calcium channel is modified by attaching the nanobody sequence to its intracellular N-terminal which then tethers the ferritin aggregate. This brings the heating component (ferritin bound iron) and the effector component (TRPVl) of NICE together at the cell surface.
  • TPRV1 temperature dependent calcium channel
  • ferritin complex There are 4 forms of the ferritin complex that have been tested - ferritin light chain-linker-heavy chain (ferritin complex) expressing in the cytoplasm, ferritin complex expressed at the cell membrane, ferritin complex with egfp fusion that binds to TRPV1 with anti-gfp camelid antibody fusion and the system described above.
  • Figure 15 demonstrates proinsulin release from 293T cells transfected with calcium dependent insulin and either TRPVl/myristoylated ferritin, camelid anti-GFP fused to TRPVl/EGFP ferritin or camelid anti-GFP fused to TRPVl/EGFP ferritin/camelid anti-GFP fused to ferritin in response to RF treatment.
  • nanoparticles are bound to the cell surface, the proportion of heat generated by Brownian motion is significantly decreased. In contrast to other particles, such as cobalt ferrite nanoparticles, heat generated by iron oxide particles is primarily through Neel fluctuation rather than Brownian motion (Fortin et al., 2007 J. Am. Chem Soc. 129:2628-2635). Thirdly, functionalized iron oxide nanoparticles are readily available in a number of sizes allowing the heating response to be tuned through field strength and frequency, and particle size. The size of particles that diffuse freely in a number of in vitro and in vivo settings has been defined and 20-30 nm beads have been found to be optimal.
  • Iron oxide nanoparticles coated with streptavidin were used to decorate specific cell populations by expression of a tether in the form of a biotin acceptor protein (BAP) fused to the transmembrane domain of PDGFR and tagged with hemagglutinin (HA) (Tannous et al, 2006 Nat Methods 3:391-396)
  • BAP biotin acceptor protein
  • HA hemagglutinin
  • the biotin acceptor protein is modified by endogenous biotin ligase.
  • the affinity of streptavidin and biotin is 10-15M and this high affinity has been shown by us to lead to specific binding of the nanoparticles only to cells that express the BAP tether.
  • nanoparticles can decorate transfected HEK293t cells that express an HA tagged BAPTM fusion protein (Figure 23). Electron microscopy confirms that streptavidin-coated ferrous oxide nanoparticles bind to the cell membrane and quantification reveals that this is significantly greater on cells transfected with the BAPTM fusion protein versus cells not expressing this construct. In addition, immunocytochemistry demonstrates significant co-localization of TRPVl with HA (BAP) immunoreactivity, TRPVl with biotin immunoreactivity, and TRPVl with Alexa 594-streptavidin binding. These results confirm that constructs can drive co-expression of TRPVl and the BAPTM fusion protein in the same cells ( Figure 23).
  • TRPVl channel This is a single component channel that detects small changes in temperature just above the physiological range (>42°C) as a result of a conformational change allowing calcium entry (Caterina et al., Nature 389:816-824).
  • the response is proportional to the temperature change and relatively rapid, thus transducing temperature variations induced by the heated nanoparticles into a graded calcium current with attendant cell activation. Repeated heating also potentiates calcium entry, i.e., there is no desensitization.
  • ion channels such as TRPVl offer many advantages for modifying cell activity, including as described above, their fast temporal resolution and the ability to target their expression genetically without the tissue damage often seen with direct electrical stimulation.
  • the activation of a temperature sensitive channel provides the same advantages as the reported light activated cation channel but without the need for an indwelling catheter, and with the potential, by using different nanoparticles, to effect activation of different cell types in response to different RF frequencies to modulate multiple cells in the same local region.
  • NICE can induce calcium entry in response to radio waves using streptavidin-coated iron oxide nanoparticles, expression of a cell surface biotin acceptor protein and the temperature sensitive TRPVl channel in transfected HEK293t cells both with calcium imaging and a calcium sensitive NFAT-luciferase reporter construct as the readout (Figure 24).
  • the calcium response can be modulated both by the power of the RF field and by the exposure time.
  • NICE in vitro can be extended as follows: (i)examining the cellular responses to nanoparticles over time, (ii) validating the ability of NICE to stimulate hormone release in vitro, (iii) modulating neural activity in vitro, in hippocampal neurons or (iv) dopaminergic neurons in brain slices and (v) examining the combinatorial activation of distinct cell populations using a novel combination of epitope-tagged TRPV1 channel and functionalized nanoparticles tuned to heat at distinct frequencies.
  • Nanoparticles to be used in these studies will be prepared using well described methods and optimized to reduce nonspecific binding by use of hydrophobic particles and functionalization with polyethylene glycol.
  • Iron oxide nanoparticles will be synthesized using iron (III) acetylacetonate, reduced in the presence of oleylamine and oleic acid to yield monodispersed and single crystalline Fe 3 0 4 nanoparticles with a high magnetic moment (Xu Zea 2009 Chem Mater 21 : 1778-1780
  • the resulting hydrophobic nanoparticles then are functionalized with carboxyl-terminated polyethylene glycol (PEG-COOH), which will further covalent attachment of targeted biomolecules (streptavidin or antibodies) through EDC/NHS chemistry and reduce nonspecific uptake by cells (Xie Jea 2007 Adv Mater 19:3163-3166)
  • Gold nanoparticles will be prepared by a seed-mediated growth synthesis (Sau T, Murphy C 2004 Langmu).
  • Nanoparticle characterization will take place through transmission electron microscopy (TEM), dynamic light scattering, X-ray diffraction and, in the case of gold, surface plasmon resonance to ensure
  • NICE utilizes cell surface tethering of nanoparticles for cell specificity and to provide proximity for TRPV1 channel activation.
  • the fate of the nanoparticles after binding will be examined using both quantitative and qualitative tools by inductively coupled plasma mass spectrometer (ICP-MS) and microscopy, respectively.
  • ICP-MS inductively coupled plasma mass spectrometer
  • HEK 293t cells transfected with TRPVl and BAPTM will be incubated with the nanoparticles and, at 5 min, 30min, 2h, 4h, 12h and 24h the cells will be washed with Tween 20 to remove surface-attached
  • NICE The ability of NICE to modify endogenous insulin gene expression, synthesis and release will first be assessed using the RIN m5F insulinoma cell line expressing TRPVl - BAPTM in vitro.
  • the stably transfected cells will be incubated in Kreb's phosphate buffer (KRB) containing streptavidin coated 20 nm iron oxide nanoparticles for 30 min.
  • Nanoparticle decorated cells will be exposed to the 465kHz RF field and the effects will be assessed after 5 min, 10 min, 30 min and 2h, in part guided by the time course studies described above. Additional studies will examine the effect of repeated pulsed RF exposure for 5, 10 or 20 min periods for a total period of 2 h.
  • Calcium imaging will be used to determine the time course of calcium entry and calcium induced insulin release into the media will be measured by ELISA with insulin gene expression examined by qPCR. The effects of RF field strength and duration on insulin secretion will be examined. Control studies will examine the effect of TRPVl and BAPTM expression on insulin secretion in the absence of RF stimulation and the effects of RF stimulation without addition of nanoparticles.
  • Calcium influx into cells can regulate gene expression via calcium dependent response elements. To assess the ability of NICE to modify gene expression, cells are engineered to express and release insulin in a Ca2+ dependant manner.
  • NFAT Nuclear Factor of activated T-cell
  • This NFAT- TATA promoter is used to drive a bicistronic construct with modified human insulin and luciferase expression ( Figure 1). Processing of proinsulin to insulin relies on two beta-cell specific prohormone convertases, so a modified human proinsulin with engineered furin cleavage sites is used as this is processed to mature insulin in non beta-cells both in vitro and in vivo (Shifrin AL, et al.
  • NICE NICE to induce insulin expression and release
  • Cells are incubated with streptavidin iron oxide nanoparticles for the final 30 min of the preincubation period. The cells are then washed and placed in a 465kHz RF field and the effects on insulin expression and release are examined by qPCR and ELISA, respectively. The effects of RF field strength and duration on gene expression and insulin release are examined and optimized.
  • TRPV1 and NFAT insulin confirm the ability of NICE to induce insulin gene expression and hormone release. See Figure 8 and Figure 6, respectively.
  • NICE Effect of NICE on Electrically Excitable Cells In Vitro. Calcium entry into electrically excitable cells induces depolarization and initiates action potentials. NICE will be used to regulate the activity of electrically excitable neurons in culture and in tissue slices in vitro. The ability of NICE to induce neural activity will be examined in primary hippocampal cell cultures. These cells will be infected with recombinant adenovirus expressing TRPV1- BAPTM constructs and then incubated with streptavidin-coated nanoparticles. Nanoparticle decorated cells will be exposed to the RF field and the effects assessed by calcium imaging and whole cell patch clamping. Preliminary studies indicate electrophysiological recording in the electromagnetic field is feasible.
  • TRPVl and the beta actin promoter return to the correct orientation and are locked in place by the excision of the additional lox site.
  • This system will allow cell specific expression of the TRPVl and BAPTM in cells that express ere with little or no expression in cells that do not express ere.
  • Transgenic mice expressing ere recombinase in dopamine transporter neurons DAT-cre will be crossed to a reporter strain, ROSA26-lox-stop-lox-EYFP mice (Ekstrand MI, et al.
  • DAT-cre/YFP mice (age 4 weeks) will receive a stereotactic injection of high-titer Ad-FLEX-NICE stocks ( ⁇ 150 nl of stock of 108 infectious units) into the ventral tegmental area (VTA).
  • expression of the construct will be determined by triple IHC to confirm co-localization of YFP (expressed solely in ere neurons), TRPVl and Alexa-streptavidin binding. Wild-type mice also will be injected to ensure there is no expression of the ere dependent construct in the absence of ere recombinase.
  • mice Ten to 14 days after viral delivery, mice will be sacrificed and sections (200 ⁇ ) will be cut. After resting, slices will be incubated with streptavidin- coated iron oxide nanoparticles for 30 min. The electrophysiological properties of infected, decorated neurons will be determined by patch clamp recording in the absence and presence of RF stimulation. Overall, these studies will confirm the ability of NICE to stimulate Ca2+ influx and neural activity in neural slices in vitro.
  • Nanoparticles of different compositions and shapes heat at different EM power and frequency with different rates. For example, iron oxide nanoparticles are heated by a frequency of 465kHz which does not heat gold, whilst gold nanoparticles heat rapidly at a frequency of 13.5MHz. Furthermore the response characteristics of the particles also are influenced by their size. Thus, gold could be activated at a much lower RF field strength than iron oxide (i.e., gold heating faster than iron oxide) (Saleh S. et al.J.
  • TRPV1 channels expressed under the control of cell- specific promoters provide distinct cell surface tags to direct nanoparticle binding.
  • Particles can be specifically targeted to different cell types by coating them with recombinant antibodies directed against short epitopes. This can allow differential regulation of distinct cell populations in the same anatomical region.
  • Specific antibodies recognizing a library of short linear epitopes that have been identified using phage display will be used. These epitopes will be inserted in frame into the first extracellular loop of TRPV1 which has been shown can be modified without altering the response characteristics of this channel.
  • Each construct will be transfected into HEK 293t cells and paired with a distinct NFAT-RE driven luciferase reporter (CBRluc - red luciferase, CBG991uc - green luciferase) to give two different reporter read-outs of calcium entry for each cell type.
  • CBRluc - red luciferase CBG991uc - green luciferase
  • CBG991uc - green luciferase NFAT-RE driven luciferase reporter
  • the localization of different particles to different cells will be confirmed using IHC.
  • the cells will then be exposed to sequential pulses (5 min, 10 min or 20min) of RF frequencies of 465kHz andl3.5MHz using a variable frequency amplifier for a total of 40 minutes at each frequency before the cells are lysed and individual luciferase activity measured.
  • stably transfected cells will be injected subcutaneously into nude mice to create tumors expressing TRPVl, BAPTM, and NFAT -insulin IRES luciferase constructs. This will be confirmed by IHC. Freely diffusable iron oxide nanoparticles (20 nm) or vehicle will be injected into the tumor and the effects of RF stimulation on blood glucose and insulin, and glucose tolerance will be examined. Insulin expression will be determined by qPCR of tumor RNA in a subset of mice. Luciferase expression, as an indicator of calcium entry, in tumors will be confirmed by luminometer studies in vivo (Birsoy K.
  • mice When these parameters have been optimized, mice also will undergo glucose tolerance testing in the presence or absence of RF stimulation. Pending these results, these studies will be repeated on control and InsTRPVl -BAPTM mice treated with streptozocin (a model of insulin deficient diabetes), high fat diet (a model of insulin resistant diabetes) or transgenic mice crossed to leptin deficient, glucose intolerant, ob/ob mice.
  • Combinatorial Activation of Nanoparticles to Regulate Glucose Metabolism In Vivo can be applied to develop a system whereby circulating glucose can be modulated through the release of either insulin or glucagon.
  • PC 12 are stably trans fected to give two populations, the first expressing TRPV1 modified with a His6x tag and NFAT driven insulin expression (PC12-TRPVlHis-Ins) and the second expressing TRPV1 modified with a FLAG tag and NFAT driven glucagon expression (PC12-TRPVlFLAG-Glucagon).
  • a combination of the two cell lines are then injected subcutaneously into nude mice to give a tumor with a mixed cell population of both PC12-TRPVlHis-Ins and PC12-TRPVlFLAG-Glucagon which can be confirmed by IHC for His and Flag epitopes.
  • the tumor is then injected with a cocktail of iron oxide nanoparticles functionalized with anti-His6x antibody and gold nanoparticles functionalized with anti- FLAG antibody.
  • RF stimulation at 465kHz should heat iron oxide nanoparticles and stimulate PC12-TRPVlHis-Ins cells to synthesize and release insulin reducing plasma glucose since gold nanoparticles are not heated at this lower frequency.
  • RF stimulation at 13.5MHz preferentially heats gold nanoparticles targeted to activate PC12-TRPVlflag- Glucagon cells to release glucagon and increase plasma glucose.
  • alternating 10 minute pulses of RF frequencies of 465kHz andl3.5MHz using a variable frequency amplifier will be used to stimulate the release of both glucagon and insulin to modulated plasma glucose.
  • VTA ventral tegmental area
  • mice with dopamine neuron specific expression of ChR2 show a significant increase in consumption from the light-activating lickometer port ( Figure 28).
  • This paradigm provides a means for comparing the robustness of light vs. RF for neural activation of this reward behavior.
  • ChR2 injected and control mice will be implanted with a fiber optic system to deliver blue light (473 nm wavelength) to the VTA and allowed to recover prior to training.
  • NICE mice and a second group of control mice will receive a VTA injection of streptavidin-coated iron oxide nanoparticles. The location of the nanoparticle injection will be confirmed by MRI, which can be used to precisely localize the beads. The distribution of particles on dopaminergic neurons will also be analyzed using double IHC for streptavidin and dopamine.
  • mice Following acclimation to the behavioral equipment (Med Associates), mice will be given three daily 60min training sessions to establish the association between one of two lickometers (L-A or L-B; counterbalanced) and intracranial stimulation either via a pulse of blue light (ChR2) or a pulse of RF field (NICE). For half the animals, L-A will be rewarded and L-B unrewarded and for half, L-B will be rewarded and L-A unrewarded. Consumption from the lickometer at each training session and at testing will be recorded by the infrared monitoring system. Having acquired the association between the lickometer and delivery of light or RF field, retrieval will be tested 2 days after training.
  • mice will be sacrificed and specific expression of TRPV1-BAPTM (anti-TRPVl, anti- tyrosine hydroxylase (TH) and Alexa-streptavidin) or ChR2 (mcherry, anti-TH) in the DAT neurons confirmed by immunohistochemistry.
  • the RF field will heat metal and therefore the behavioral equipment must be free of all metal components. This will be achieved by using a custom- built plexiglass chamber with glass sippers and fiber optical cabling delivering information from the infrared beam break system. A video system mounted above the RF field will monitor movement in the chamber. The chamber will be encircled by a custom-made coil sufficient to provide a uniform RF field throughout the chamber and to a height equivalent to that of a rearing mouse.
  • NICE is most advantageous for the study of complex behaviors where intracranial implants and tethering to deliver light or electrical stimuli may subtly alter or hinder behavior.
  • One such behavior is feeding, which is particularly susceptible to interference such that even relatively low levels of stress may alter feeding patterns (Abbott CR et al. 2006 Int J Obes (Lond) 30:288-292).
  • the hypothalamus regulates feeding to maintain body weight in a narrow range while hedonistic systems, particularly dopamine reward pathways and cortical regions, modulate and may even over-ride this (Davidson TL 1993 Psychol Rev 100:640-657).
  • the hypothalamic arcuate nucleus is known to be a key component of hypothalamic feeding circuitry.
  • the arcuate contains two primary cell populations expressing the leptin receptor, one co-expressing neuropeptide Y (NPY) and agouti related peptide (Agrp) and the second co-expressing the gene pro-opiomelanocortin (POMC) whose major product is alpha melatonin stimulating hormone (a-MSH) (Cone RD, 2001 Int J Obes Relat Metab Disord 25 Suppl 5:S63-S67)
  • a-MSH alpha melatonin stimulating hormone
  • Central and arcuate Agrp injection potently stimulates feeding, knock-down in Agrp expression reduces body weight and postnatal loss of Agrp neurons reduces food intake and body weight (Bewick GA et al.
  • these neurons also express classical neurotransmitters and other neuropeptides whose effects may not be reflected in the pharmacological approaches of intracranial peptide delivery or peptide over-expression.
  • the relative dominance of these neural populations on feeding is unknown for example, whether stimulation of Agrp neurons outweighs the effect of stimulating POMC neurons or vice versa.
  • NICE will be used to selectively stimulate these important neuronal populations in vivo, either individually or together, to examine their physiological roles in feeding.
  • BAC transgenesis will be used to generate mice expressing epitope-tagged TRPV1 channels directly under the control of the Agrp promoter (His tagged) or the POMC promoter (FLAG tagged) ( Figure 11) (Gong S et al. 2002 Genome Res 12: 1992-1998). When these mice are crossed, dual transgenic progeny expressing Agrp-TrpvlHis and POMC- TrpvlFLAG will be generated ( Figure 29C). Specific expression will be confirmed by dual IHC for His/ Agrp and FLAG/a-MSH. Mice will receive a stereotactic injection into the arcuate nucleus of a mixture of anti-His coated iron oxide nanoparticles and anti-Flag gold nanoparticles.
  • a combination of nanoparticle heating in an RF field with defined expression of a cell surface tag for targeted nanoparticle binding will be used together with a temperature responsive ion channel to convert localized temperature changes into remote, temporally controlled and anatomically defined cell activation.
  • a temperature responsive ion channel to convert localized temperature changes into remote, temporally controlled and anatomically defined cell activation.
  • ion channels has many advantages; their structure and function are relatively well described, they have a rapid time course of activation and a broad range of channels exist in mammalian and non-mammalian cells, which may be exploited in the search for the optimum means of modifying cellular activity.
  • the non-mammalian channelrhodopsin (ChR2) gene a light activated cation, has been employed to rapidly activate molecularly defined neurons when exposed to blue light.
  • This system gives anatomical specificity and temporal control but requires fiber optic light delivery via invasive chronic implanted devices because light penetration is relatively poor.
  • a means for activating ion channels without the requirement of an implanted device could represent an important potential means for activating cells.
  • Describe herein is a means for non-invasive excitation of a defined cell population in vivo using a radiofrequency field to heat iron oxide nanoparticles which in turn activate the temperature sensitive TRPV1 channel to trigger calcium entry. Calcium influx in turn activates gene expression and in the current system, hormone release. This approach has been validated by modulating the expression and secretion of the peptide hormone, insulin, and shown that it can be used to lower blood glucose in mice.
  • Metal nanoparticles can readily be functionalized to target to defined cell populations by coating with specific antibodies that recognize proteins that are normally expressed on a cell or transfected into that cell. Thus, nanoparticles are well suited for inducing cell surface temperature changes that can be transduced into cellular responses by temperature sensitive channels in vitro and in vivo.
  • TRPVl channel The local temperature change achieved by exposing nanoparticle-coated cells to radio waves can be transduced into calcium entry by targeted expression of TRPVl channel.
  • This single component, cell surface cation channel detects small changes in temperature just above the physiological range (>42°C) and undergoes a conformational change allowing calcium entry.
  • the response is proportional to the temperature change and relatively rapid, thus transducing temperature variations induced by the heated nanoparticles into a graded calcium current with attendant cell activation.
  • these channels do not desensitize after repeated activation as repeated heating potentiates calcium entry.
  • the method described herein makes use of metal nanoparticles, directed to specific cells, and TRPVl ion channels to remotely stimulate cell activity and gene expression as follows.
  • a single construct encoding TRPVl, modified to incorporate a unique extracellular His x 6 epitope tag was expressed in specific cells (TRPVl His).
  • Iron oxide (IO) nanoparticles functionalized with monoclonal antibodies against the His x 6 epitope tag are targeted to these cells.
  • IO Iron oxide
  • VI BAP transmembrane protein as a nanoparticle anchor and a separate TRPVl to induce calcium entry
  • Functionalized iron oxide nanoparticles were used to decorate the cells. Size dependent heating of IO nanoparticles occurred at 465kHz with maximum heating achieved using an aqueous solution of 20nm diameter particles. With an alternating magnetic field (465kHz,), an increase of up to 17°C can be achieved in a suspension of 20nm iron oxide particles (1 mg/ml) (Figure 2A) with an initial heating rate of 0.15°C/s. A 5°C rise in nanoparticle temperature above body temperature is sufficient to open TRPVl channels and can be reached in 30s. Nanoparticles of this size are within the limits of the extracellular space and therefore able to diffuse.
  • IO nanoparticles (20nm) functionalized with monoclonal anti-His antibody bind to TRPVl His expressed in transfected human embryonic kidney (HEK 293) cells.
  • TRPVlHis (19.78 ⁇ 2.23 nanoparticles per mm cell membrane)
  • untransfected cells (2.85 ⁇ 0.32 nanoparticles per mm cell membrane)
  • Figure 30A and B the density achieved with streptavidin functionalized IO nanoparticles bound to cells expressing the two component system, TRPVl BAP (9.07 ⁇ 2.85 nanoparticles per mm cell membrane).
  • Nanoparticle activated insulin expression and release in vitro In order to assess the potential anti-diabetic effects of this system, the modified TRPVl construct and an NF AT -insulin construct was introduced into PC 12 cells as well as 293T cells. These cells process and secrete proteins in response to increased intracellular Ca++.
  • a synthetic promoter comprised of three calcium response elements: serum response element (SRE), cyclic AMP response element (CRE) and nuclear factor of activated T-cell response element (NFAT RE) and a minimal promoter were cloned upstream of a modified, furin sensitive insulin cDNA (Figure 19A).
  • proinsulin to insulin relies on two beta cell specific prohormone convertases so a modified human proinsulin with engineered furin cleavage sites was used.
  • This expressed protein is processed to mature insulin in non beta cells both in vitro and in vivo (Shifrin et al., gene Ther. 8: 1480-1489) and its release can be differentiated from endogenous murine insulin release in vivo.
  • beta cells expressing furin sensitive insulin synthesize both proinsulin and insulin and the ratio of proinsulin to insulin varies both with cell type and basal vs activated state.
  • TRPVlHis or TRPVl BAP were decorated with functionalized IO nanoparticles. Applying a RF magnetic field to nanoparticle-decorated cells expressing TRPVlHis or TRPVl BAP and calcium regulated furin sensitive insulin significantly increased proinsulin release (Figure 19B) (TRPVlHis: control 100 ⁇ 13.94 %basal, RF treated 671 ⁇ 234.9 %basal. TRPVl BAP: control 100 ⁇ 23.96 %basal, RF treated 477.4 ⁇ 136.1 %basal) and insulin gene expression (Figure 19C) (TRPVlHis: control 1.0 ⁇ 0.18, RF treated 2.20 ⁇ 0.53 relative gene
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
  • Nanoparticle regulation of blood glucose in vivo Experiments were conducted to translate in vitro findings in vivo by testing whether the remote activation of proinsulin gene expression, insulin synthesis and release could modulate blood glucose in mice. Stably transfected PC 12 cells expressing TRPVl His and calcium regulated furin sensitive insulin were established. As above, this endocrine cell line is capable of synthesizing and secreting mature insulin. In vitro studies examining the effects of RF treatment on proinsulin release and insulin gene expression replicated the findings in transfected HEK 239t cells ( Figure 33 A - C).
  • Stably transfected PC12-TRPVlHis-Ins cells were injected subcutaneously into the flank of nude mice and formed tumors expressing TRPVlHis ( Figure 33D) and furin sensitive insulin constructs. Following an overnight fast, PBS or 10 nanoparticles were injected into the tumors of anesthetized mice (50ul total volume, nanoparticle concentration 8mg/ml). Blood glucose and plasma insulin were measured before, during and after the application of an RF field (See Figure 33E).
  • Plasma insulin was also significantly increased in mice receiving nanoparticle injection and RF magnetic field treatment but remained unchanged in control mice injected with PBS (Figure 10C)(Plasma insulin: PBS (-30min) 1.83 ⁇ 0.38 ⁇ / ⁇ 1, PBS (30min) 1.75 ⁇ 0.36 ⁇ / ⁇ 1, nanoparticles (-30min) 2.26 ⁇ 0.76 ⁇ / ⁇ 1, nanoparticles (30min) 3.25 ⁇ 0.64 ⁇ / ⁇ 1, p ⁇ 0.05).
  • proinsulin mRNA levels were significantly higher in nanoparticle injected tumors treated with RF magnetic field compared to those injected with nanoparticles alone (Figure 10D) (Control: 1.0 ⁇ 0.2 relative insulin gene expression, RF treated: 2.03 ⁇ 0.3 relative insulin gene expression, p ⁇ 0.05). There was no difference in apoptosis, assessed as before by TUNEL and activated caspase-3, between nanoparticle injected tumors in the presence or absence of the RF magnetic field (Figure 10E and F)(TUNEL: control 56.4 ⁇ 9.8 cells, RF 48.8 ⁇ 8.9 cells. Active Caspase 3: control 199.8 ⁇ 27.4 cells, RF 168 ⁇ 24.4 cells)
  • the system described herein can be further modified to achieve combinatorial activation of different cells.
  • Cell populations expressing TRPV1 engineered to incorporate unique epitope tags could be decorated with different nanoparticles designed to heat in response to distinct RF frequencies allowing combinatorial cell activation and peptide release.
  • gold nanoparticles could be labeled with a second antibody to a different epitopes on the modified TRPV1 channel in cells engineered to express glucagon. This would enable one to either lower or raise blood glucose depending on the ambient blood glucose concentration.
  • These studies provide a platform for developments using different types of nanoparticles with enhanced characteristics.
  • nanoparticle structures could be used to depolarize cells without the need for a TRP channel; this advance would allow the direct activation of cells using nanoparticles with antibodies directed against specific cell surface epitopes. It may also be possible to engineer cells that express nanoparticles intracellularly thus obviating the need for injecting particles.

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Abstract

La présente invention concerne des méthodes et des compositions permettant de réguler à distance la fonction cellulaire grâce au recours à des ondes radiofréquence pour exciter des nanoparticules ciblant des types cellulaires particuliers. Lesdites nanoparticules peuvent être appliquées sur la cellule cible de manière extracellulaire et/ou exprimées de façon intracellulaire. Le type cellulaire considéré exprime un canal sensible à la température dans lequel l'excitation des nanoparticules entraîne une augmentation localisée de la température qui est transduite en une réponse cellulaire. Lesdites réponses cellulaires peuvent comprendre, par exemple, une augmentation de l'expression génique entraînant la production d'une ou plusieurs protéines physiologiquement actives. L'expression desdites protéines peut être utilisée pour traiter diverses maladies ou affections héréditaires ou acquises chez un sujet. En conséquence, l'invention concerne une démarche générique de traitement d'une quelconque maladie associée à une déficience en protéines.
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